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Abstract

Forster resonance energy transfer (FRET) is a nonradiative transfer of energy between two
fluorescent molecules (a donor and an acceptor) in nanometer range proximity. FRET imaging
methods have been applied to proteomic studies and drug discovery applications based on
intermolecular FRET efficiency measurements and stoichiometric measurements of FRET interaction
as quantitative parameters of interest. Importantly, FRET provides information about
biomolecular interactions at a molecular level, well beyond the diffraction limits of standard
microscopy techniques. The application of FRET to small animal imaging will allow biomedical
researchers to investigate physiological processes occurring at nanometer range in vivo as well
as in situ. In this work a new method for the quantitative reconstruction of FRET measurements
in small animals, incorporating a full-field tomographic acquisition system with a Monte Carlo
based hierarchical reconstruction scheme, is described and validated in murine models. Our main
objective is to estimate the relative concentration of two forms of donor species, i.e., a
donor molecule involved in FRETing to an acceptor close by and a nonFRETing donor molecule.

a, Design of murine phantom with four inclusions carrying mixtures with different acceptor
to donor ratios (Red- 1:4, Green-1:2, Cyan-2:1 and Blue-4:1). The orange boundary indicates
the area of full-field excitation. b, An example of the normalized temporal measurements
acquired at detectors directly above the four inclusions when excited by a full-field
excitation pattern. c, Histogram of the value of shorter lifetime (FRETing donor) component
for all detectors above signal threshold for all excitation patterns.

a, 3D rendering of the CT images of the mouse model showing the location of two capillary
tubes carrying donor acceptor mixtures with A:D ratio of 1:4 (green) and 4:1 (blue). The
black border represents the registered field of view on the optical imaging platform. b, An
example of the temporal measurements acquired at detectors directly above the four
inclusions when excited by a full-field excitation pattern. c, Histogram of the value of
shorter lifetime (FRETing donor) component for all detectors above signal threshold for all
excitation patterns.